This prospective, randomized, open-label study was registered in the Chinese Clinical Trial Registry Centre (Registration No. ChiCTR1900021269) and approved by the Medical Ethics Committee of Sichuan Provincial Hospital for Woman and Children. Signed informed consents were obtained from all participants. All procedures in this study complied with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration 1975 (2013 revision).
The sample size calculation was based on the assumption that the clinical pregnancy rate would increase threefold after GH pretreatment. The clinical pregnancy rate of the poor ovarian responders in our centre was approximately 13%, and 48 patients were required in each group, with an α of 0.05 and a β error of 0.1 (power = 90%)
The patients with POR (aged 33-43 years) diagnosed according to the Bologna criteria , who underwent IVF, were enrolled from the Reproductive Medicine Centre of Sichuan Provincial Hospital for Women and Children (between Feb. 2019 and Dec. 2019). The poor ovarian responders were randomized 1:1 to either GH pretreatment (the POR-GH group) or no GH pretreatment (the POR-C group) (using computer-generated random numbers). The exclusion criteria were as follows: (1) hydrosalpinx, congenital uterine malformations and/or endometrial disease such as tuberculosis or hyperplasia; (2) basal follicle-stimulating hormone (bFSH) level ≥15 IU/L; (3) systemic lupus erythematosus, sicca syndrome, or polycystic ovarian syndrome; (4) an uncontrolled endocrinopathy such as diabetes, hyperthyroidism, hypothyroidism, or hyperprolactinemia; (5) IVF-ET treatment within three months; (6) an intracytoplasmic sperm injection (ICSI) cycle due to male infertility; or (7) supplementation with any antioxidants such as vitamin E, vitamin C, CoQ10, beta-carotene or selenium. Women with tubal factor infertility (aged 20-35 years) with normal ovarian reserve and regular menstrual cycles who underwent IVF-ET were recruited as non-POR controls during the same period. The exclusion criteria for the non-POR group were the same as those for the POR group.
Data involving the medical history of each participant were collected and included the regularity of menstrual cycles, duration of infertility, and history of treatment. Abnormal menstrual cycles included oligomenorrhoea, polymenorrhoea, irregular menstrual cycles, and amenorrhoea. Body mass index (BMI) was calculated as body weight divided by the square of body height (kg/m2). The antral follicle count (AFC) was determined by transvaginal ultrasound on days 2-3 of menstruation or progestin-induced bleeding withdrawal.
Controlled ovarian stimulation (COS) and IVF
All patients underwent controlled ovarian stimulation (COS) with the GnRH antagonist protocol. Recombinant follicle-stimulating hormone (rFSH) (Gonal-F; Merck-Serono KGaA., Darmstadt, Germany) was injected on day 2 of the menstrual cycle, and rFSH doses were adjusted according to follicle growth and serum hormone levels. In the POR-GH group, 4 IU/d recombinant human growth hormone (Changchun GeneScience Pharmaceuticals Co., Ltd., Changchun, Jilin, China) was injected subcutaneously on day 2 of the previous menstrual cycle before IVF until the trigger day (36-48 days). When a leading follicle reached 12 mm and/or serum E2 levels reached 300 pg/mL, Ganirelix (Merck Sharp & Dohme co., Ltd, Hoddesdon, United Kingdom) was administered. When at least one follicle was above 18 mm, recombinant human chorionic gonadotropin (hCG) (Ovitrelle®; Merck-Serono KGaA) was administered as the trigger. When there were no follicles with diameters ≥14 mm after 10 days of gonadotrophin injection or when the peak E2 level was below 300 pg/mL, the cycle was cancelled. Ultrasound-guided transvaginal oocyte retrieval was performed 36 hours later, and follicle flushing was not performed. Upon oocyte retrieval, FF was collected only from follicles with a diameter ≥16 mm measured on the retrieval day. FF samples were immediately centrifuged at 700 g for 5 min at room temperature, and the supernatant was stored at -80°C. The precipitates were suspended in 3 ml PBS, gently layered in 3 ml 50% Lymphocyte separation medium (Beijing Solarbio Science and Technology Corporation, Beijing, Solarbio Science and Technology Co., Ltd, China) and then centrifuged at 700 g for 10 min to remove red blood cells and debris. GCs layered at the interface of the gradient were washed twice with 5 ml PBS (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, Jiangsu, ChinaKeyGEN Bio TECH Co., Ltd., Jiangsu, China) and immediately examined for ROS using ﬂuorescence microscopy and spectrophotometry. FF or GCs from each patient were collected separately and considered to be one sample. According to the criteria established by the Istanbul Consensus Workshop on Embryo Assessment, the cultured embryos on day 3 were assessed based on the number of blastomeres and the degree of fragmentation, and higher quality embryos were categorized as grade A/B . One or two fresh embryos with the best morphological grade were selected for transfer. The remaining embryos were all cultured to the blastocyst stage to be frozen. Luteal phase support with intramuscular injection of progesterone 60 mg/d commenced on the oocyte retrieval day. Serum hCG was measured 12 days after ET, and hCG positivity was considered when hCG≥5 IU/mL. Clinical pregnancy was defined as demonstration of a gestational sac with an embryo showing cardiac activity. Early miscarriage was deﬁned as loss of pregnancy before gestational week 12. The implantation rate was calculated as the observed number of gestational sacs divided by the number of transferred embryos. The clinical pregnancy rate was expressed as the number of clinical pregnancy cycles divided by the number of embryo transfer cycles. The miscarriage rate was expressed as the number of spontaneous pregnancy loss cycles before 12 weeks of gestation divided by the number of clinical pregnancy cycles. Ovarian hyperstimulation syndrome (OHSS) was diagnosed according to Navot D et al .
We suggest GH 4 IU/d pretreatment on day 2 of the previous menstrual cycle before IVF until the trigger day because a low physiological dose and longer treatment (from the antral follicle stage) might be more beneﬁcial to follicular growth and development .
Measurement of endocrine and metabolic parameters
Plasma glucose was measured using the hexokinase method (Beijing Strong Biotechnologies, Inc., Beijing, China). Oestradiol (E2), progesterone (P), total testosterone (TT), luteinizing hormone (LH), FSH, and insulin levels were measured using the electrochemiluminescence immunoassay platform (Roche Diagnostics GmbH, Mannheim, Germany). The serum level of anti-Mullerian hormone (AMH) was measured using an enzyme-linked immunosorbent assay kit (Guangzhou Kangrun Biotech, Co., Ltd, Guangdong, China). The homeostasis model assessment (HOMA-IR) index was calculated as fasting glucose (mmol/l) × fasting insulin (mU/mL)/22.5 . The intra- and inter-assay coefficients of the above variation were less than 5% and 10%, respectively.
OS marker in FF assay procedures
FF malondialdehyde (MDA) concentrations (µmol/L) were measured using micro-MDA detection kits (NanJing Jiancheng Bioengineering Institute Co. Ltd., Nanjing, Jiangsu, China) and ultraviolet spectrophotometry (Shanghai Meipuda Instrument Co., Ltd., Shanghai, China) was performed at 532 nm. Superoxide dismutase (SOD) was determined using SOD kits (NanJing Jiancheng Bioengineering Institute Co. Ltd.) and spectrophotometry at 450 nm. Total antioxidant capacity (TAC) (mmol Trolox Equiv./L) and TOS (µmol H2O2 Equiv./L) were measured by the semiautomatic microplate colorimetric method using hydrogen peroxide (H2O2) and Trolox, respectively, for the standard curve [22,23]. The oxidative stress index (OSI) was expressed as the ratio of TOS to TAC. The measurements were performed in duplicate for each OS parameter. Serum samples from healthy volunteers were pooled for quality control. The intra- and inter-assay coefficients of the above variations were less than 5% and 10%, respectively.
Detection of intracellular ROS levels
With a dichlorodihydroﬂuorescein diacetate (DCFH-DA) ﬂuorescent probe, ROS levels in GC were detected by an ROS assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). Briefly, GCs were resuspended in PBS and incubated with 10 µmol/L DCFH-DA in the dark for 25 min (37°C) and then incubated together with 10 µg/mL 4',6-diamidino-2-phenylindole (DAPI) (NeoFroxx, Frankfurt, Germany) for 5 min. After the cells were washed three times with PBS, GC suspensions were added to glass slides, and fluorescence was examined by fluorescence microscopy (Olympus Corporation, Tokyo, Japan). The examination wavelength was 488 nm, and the emission wavelength was 525 nm.
Similar with the above protocol but without DAPI, another part of GCs was used to measure the intracellular ROS level by NanoDrop UV-Vis spectrophotometry (Thermo Scientific, Massachusetts, USA). The fluorescence intensities are shown as the intensity of the POR group relative to that of the control group (non-POR group).
All data were statistically analysed using SPSS 17.0 software (SPSS Inc., Chicago IL, USA). Continuous variables were expressed as the mean ± standard deviation (SD). The Kolmogorov–Smirnov test was used to assess the normality of the data distribution. Continuous variables with normal distribution were compared using Student-Newman-Keuls test, and Bonferroni’s test was used as the post hoc test. Categorical data were compared using chi-squared tests. A two-tailed P ≤ 0.05 was considered statistically significant.